IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ Human C4b-binding protein, structural basis for interaction with streptococcal M protein, a major bacterial virulence factor. Human C4b-binding protein, structural basis for interaction with streptococcal M protein, a major bacterial virulence factor. J. Biol. Chem. (0021-9258) 2006 imex curation Jenkins HT., Mark L., Ball G., Persson J., Lindahl G., Uhrin D., Blom AM., Barlow PN. BioCreative - Critical Assessment of Information Extraction systems in Biology Paul.Barlow@ed.ac.uk 24-JAN-2007: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro itc isothermal titration calorimetry ITC ITC itc predetermined predetermined participant predetermined Jenkins HT., Mark L., Ball G., Persson J., Lindahl G., Uhrin D., Blom AM., Barlow PN. BioCreative - Critical Assessment of Information Extraction systems in Biology Paul.Barlow@ed.ac.uk Accepted 2006-SEP-7 by JYOTI. 24-JAN-2007: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation Human C4b-binding protein, structural basis for interaction with streptococcal M protein, a major bacterial virulence factor. Human C4b-binding protein, structural basis for interaction with streptococcal M protein, a major bacterial virulence factor. J. Biol. Chem. (0021-9258) 2006 imex curation Jenkins HT., Mark L., Ball G., Persson J., Lindahl G., Uhrin D., Blom AM., Barlow PN. BioCreative - Critical Assessment of Information Extraction systems in Biology Paul.Barlow@ed.ac.uk 24-JAN-2007: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro saturation binding saturation binding immunostaining immunostaining Jenkins HT., Mark L., Ball G., Persson J., Lindahl G., Uhrin D., Blom AM., Barlow PN. BioCreative - Critical Assessment of Information Extraction systems in Biology Paul.Barlow@ed.ac.uk Accepted 2006-SEP-7 by JYOTI. J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation Human C4b-binding protein, structural basis for interaction with streptococcal M protein, a major bacterial virulence factor. Human C4b-binding protein, structural basis for interaction with streptococcal M protein, a major bacterial virulence factor. J. Biol. Chem. (0021-9258) 2006 imex curation Jenkins HT., Mark L., Ball G., Persson J., Lindahl G., Uhrin D., Blom AM., Barlow PN. BioCreative - Critical Assessment of Information Extraction systems in Biology Paul.Barlow@ed.ac.uk 24-JAN-2007: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro nmr nuclear magnetic resonance NMR NMR nmr predetermined predetermined participant predetermined Jenkins HT., Mark L., Ball G., Persson J., Lindahl G., Uhrin D., Blom AM., Barlow PN. BioCreative - Critical Assessment of Information Extraction systems in Biology NMR spectra were acquired (37 �C) on Bruker AVANCE 600 and 800 MHz spectrometers. Paul.Barlow@ed.ac.uk Accepted 2006-SEP-7 by JYOTI. 24-JAN-2007: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation c4bpa_human C4b-binding protein alpha chain Proline-rich protein C4BPA C4BP protein protein human Homo sapiens Human MHPPKTPSGALHRKRKMAAWPFSRLWKVSDPILFQMTLIAALLPAVLGNCGPPPTLSFAAPMDITLTETRFKTGTTLKYTCLPGYVRSHSTQTLTCNSDGEWVYNTFCIYKRCRHPGELRNGQVEIKTDLSFGSQIEFSCSEGFFLIGSTTSRCEVQDRGVGWSHPLPQCEIVKCKPPPDIRNGRHSGEENFYAYGFSVTYSCDPRFSLLGHASISCTVENETIGVWRPSPPTCEKITCRKPDVSHGEMVSGFGPIYNYKDTIVFKCQKGFVLRGSSVIHCDADSKWNPSPPACEPNSCINLPDIPHASWETYPRPTKEDVYVVGTVLRYRCHPGYKPTTDEPTTVICQKNLRWTPYQGCEALCCPEPKLNNGEITQHRKSRPANHCVYFYGDEISFSCHETSRFSAICQGDGTWSPRTPSCGDICNFPPKIAHGHYKQSSSYSFFKEEIIYECDKGYILVGQAKLSCSYSHWSAPAPQCKALCRKPELVNGRLSVDKDQYVEPENVTIQCDSGYGVVGPQSITCSGNRTWYPEVPKCEWETPEGCEQVLTGKRLMQCLPNPEDVKMALEVYKLSLEIEQLELQRDSARQSTLDKEL 67E03F2EA85A16DD arp4_strpy IgA receptor arp4 protein protein strpy Streptococcus pyogenes MARKDTNKQYSLRKLKTGTASVAVAVAVLGAGFANQTEVKAAEIKKPQADSAWNWPKEYNALLKENEELKVEREKYLSYADDKEKDPQYRALMGENQDLRKREGQYQDKIEELEKERKEKQERQEQLERQYQIEADKHYQEQQKKHQQEQQQLEAEKQKLAKDKQISDASRQGLSRDLEASRAAKKELEAEHQKLKEEKQISDASRQGLSRDLEASREAKKKVEADLAALTAEHQKLKEDKQISDASRQGLSRDLEASREAKKKVEADLAEANSKLQALEKLNKELEEGKKLSEKEKAELQARLEAEAKALKEQLAKQAEELAKLKGNQTPNAKVAPQANRSRSAMTQQKRTLPSTGETANPFFTAAAATVMVSAGMLALKRKEEN C5FBDCDBB97BA0B8 apr4-c4bp 2 5 unspecified role unspecified role bait bait ecoli Escherichia coli (strain K12) 4 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) direct interaction direct interaction Figure 1A To define the C4BP region involved in M protein binding, deletion mutants of the -chain were tested for binding to theMprotein, apr4-c4bp-1 2 4 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 5 unspecified role unspecified role bait bait ecoli Escherichia coli (strain K12) direct interaction direct interaction Figure 1A To define the C4BP region involved in M protein binding, deletion mutants of the -chain were tested for binding to theMprotein, apr4-c4bp-2 2 4 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) 5 unspecified role unspecified role bait bait ecoli Escherichia coli (strain K12) direct interaction direct interaction Figure 1B In further investigations, the linking sequences between CCPs 1, 2, 3, and 4 of the -chain were engineered by insertions of Ala residues (Fig. 1B). Addition of residues between CCP2 and CCP3, or between CCP3 and CCP4, had no effect on binding, whereas insertions between CCP1 and CCP2 caused a loss of M4 binding. Insertion of two alanine residues between Lysine 188 and Isoleucine 189 resulted in abolished interaction between the two proteins. apr4-c4bp-3 1 4 unspecified role unspecified role neutral component neutral component c terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range n terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 5 unspecified role unspecified role neutral component neutral component n terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) direct interaction direct interaction Figure 1C A recombinant protein consisting of residues 1�124 of C4BP -chain (see Fig. 1C, C4BP12), His-tagged at the C terminus, was expressed in E. coli and refolded. ITC was used (Fig. 1D) to measure the binding affinity between C4BP12 and a dimerised peptide corresponding to the hypervariable region of M4 (residues 1�45 of M4 protein with a C-terminal Cys added. A dimer of the APR4 protein was created as previous studies had shown that dimerization strongly enhanced C4BP binding. The APR4 protein used in this stidy also had a C-terminal Cys added. apr4-c4bp-4 3 4 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 5 unspecified role unspecified role neutral component neutral component n terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) direct interaction direct interaction Figure 4 and Figure 5 Significant changes in the 15N-1H HSQC spectrum of C4BP12 occurred upon addition of M4-N The APR4 protein was present as a dimer.